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. 2019 Aug 13;9(1):11780.
doi: 10.1038/s41598-019-48162-9.

A qPCR expression assay of IFI44L gene differentiates viral from bacterial infections in febrile children

Alberto Gómez-Carballa  1   2   3 , Miriam Cebey-López  4   5 , Jacobo Pardo-Seco  4   5   6 , Ruth Barral-Arca  4   5 , Irene Rivero-Calle  4   5 , Sara Pischedda  4   5 , María José Currás-Tuala  4   5 , José Gómez-Rial  4   5 , Francisco Barros  7 , Federico Martinón-Torres  4   5 , Antonio Salas  4   5   6
Affiliations

A qPCR expression assay of IFI44L gene differentiates viral from bacterial infections in febrile children

Alberto Gómez-Carballa et al. Sci Rep. .

Abstract

The diagnosis of bacterial infections in hospital settings is currently performed using bacterial culture from sterile site, but they are lengthy and limited. Transcriptomic biomarkers are becoming promising tools for diagnosis with potential applicability in clinical settings. We evaluated a RT-qPCR assay for a 2-transcript host expression signature (FAM89A and IFI44L genes) inferred from microarray data that allow to differentiate between viral and bacterial infection in febrile children. This assay was able to discriminate viral from bacterial infections (P-value = 1.04 × 10-4; AUC = 92.2%; sensitivity = 90.9%; specificity = 85.7%) and showed very high reproducibility regardless of the reference gene(s) used to normalize the data. Unexpectedly, the monogenic IFI44L expression signature yielded better results than those obtained from the 2-transcript test (P-value = 3.59 × 10-5; AUC = 94.1%; sensitivity = 90.9%; specificity = 92.8%). We validated this IFI44L signature in previously published microarray and whole-transcriptome data from patients affected by different types of viral and bacterial infections, confirming that this gene alone differentiates between both groups, thus saving time, effort, and costs. Herein, we demonstrate that host expression microarray data can be successfully translated into a fast, highly accurate and relatively inexpensive in vitro assay that could be implemented in the clinical routine.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Boxplot of Ct raw data. Raw Ct data (y-axis) obtained from qPCR assay of candidate reference genes (x-axis) in viral, bacterial, and control cohorts. The box represents the interquartile range (25th to the 75th) containing the middle 50% of the data, the line in the box represents the median, and the whiskers represent the ranges for the bottom 25% and the top 25% of the data values, excluding outliers.
Figure 2
Figure 2
Comprehensive stability ranking results of the different candidate reference genes in the different cohorts.
Figure 3
Figure 3
Evaluation of DRS test performance. (A) Boxplot of DRS values from bacterial and viral infected samples using 1-transcript signature (IFI44L). Horizontal red dotted line indicates the optimal threshold value. (B) Boxplot of DRS values from bacterial and viral samples using 2-transcript signature. Horizontal blue dotted line indicates the optimal threshold value. (C) ROC curves generated from 2-transcript (blue) and 1-transcript (red) (IFI44L) DRS with AUC values and 95% CI. We used a t-test to evaluate statistical differences in DRS values between bacterial and viral patients in panels (A,B). The box represents the interquartile range (25th to the 75th) containing the middle 50% of the data, the line in the box represents the median, and the whiskers represent the ranges for the bottom 25% and the top 25% of the data values, excluding outliers.
Figure 4
Figure 4
Evaluation of DRS test performance in microarray data from definitive bacterial and definitive viral infection in febrile children. (A) Boxplot of DRS values using 1-transcript signature (IFI44L). (B) Boxplot of DRS using 2-transcript signature. (C) ROC curves generated from 2-transcript (black) and 1-transcript ([IFI44L; blue], [FAM89A; yellow]) DRS with AUC values and 95% CI. We used a t-test to evaluate statistical differences in DRS values between bacterial and viral patients in panels (A,B). The box represents the interquartile range (25th to the 75th) containing the middle 50% of the data, the line in the box represents the median, and the whiskers represent the ranges for the bottom 25% and the top 25% of the data values, excluding outliers.
Figure 5
Figure 5
Evaluation of DRS test performance in whole transcript data from bacterial and viral diarrhea patients. (A) Boxplot of DRS values using 1-transcript signature (IFI44L). (B) Boxplot of DRS using 2-transcript signature. Dots indicate disease severity. Dots indicate disease severity. (C) ROC curves generated from 2-transcript (blue) and 1-transcript ([IFI44L; green], [FAM89A; red]) DRS with AUC values and 95% CI. We used a t-test to evaluate statistical differences in DRS values between bacterial and viral patients in panels (A,B). The box represents the interquartile range (25th to the 75th) containing the middle 50% of the data, the line in the box represents the median, and the whiskers represent the ranges for the bottom 25% and the top 25% of the data values, excluding outliers.
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